Method of modulating alkaloid content in tobacco plants

Abstract

The present invention provides a method for modulating the alkaloid content of a plant (e.g. a tobacco plant), the method comprising modifying said plant by modulating the activity or expression of a FAD synthetase. The present invention also provides for the use of a FAD synthetase for modulating the alkaloid content of a plant, as well as tobacco cells, plants, plant propagation materials, harvested leaves, processed tobaccos, or tobacco products obtainable in accordance with the invention.

1 . A method of modulating or decreasing the content of alkaloid, a tobacco specific nitrosamine (TSNA), or a precursor of a TSNA in a tobacco plant or a part thereof or tobacco plant cell, the method comprising modifying said plant or plant cell by modulating or decreasing the activity or expression of a FAD synthetase. 
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , further comprising producing cell culture, a plant propagation material, a leaf, a cut harvested leaf, a processed leaf, or a cut and processed leaf from the tobacco plant or the part thereof or the tobacco plant cell, wherein the cell culture, the plant propagation material, the leaf, the cut harvested leaf, the processed leaf, or the cut and processed leaf has modulated or decreased alkaloid content. 
     
     
         4 . (canceled) 
     
     
         5 . The method according to  claim 1 ,
 (i) wherein the FAD synthetase:
 a) comprises an amino acid sequence as set out in SEQ ID No. 3; or a functional variant or functional fragment or orthologue of SEQ ID No. 3; or a sequence which has at least 80% identity to SEQ ID No. 3; or a homologue of SEQ ID No. 3; or 
 b) is encoded by a nucleotide sequence as set out in SEQ ID No. 1 or 2; or a functional variant or functional fragment or orthologue of SEQ ID No. 1 or 2; or a nucleic acid sequence which has at least 80% identity to SEQ ID No. 1 or 2 or a homologue of SEQ ID No. 1 or 2, or 
 (ii) wherein the alkaloid content in the tobacco plant or a part thereof or tobacco plant cell is modulated or decreased in comparison to a plant or cell culture which has not been modified to modulate the activity or expression of a FAD synthetase. 
   
     
     
         6 . (canceled) 
     
     
         7 . A tobacco plant or part thereof or a tobacco cell or cell culture, having been modified to modulate or decrease the activity or expression of a FAD synthetase, and comprising decreased alkaloid and/or TSNA precursor content in comparison to an unmodified plant or unmodified cell or cell culture. 
     
     
         8 . A plant propagation material obtained from the tobacco plant according to  claim 7 , the plant propagation material comprising modulated or decreased activity or expression of a FAD synthetase, and decreased alkaloid and/or TSNA precursor content in comparison to a plant propagation material obtained from an unmodified tobacco plant. 
     
     
         9 . (canceled) 
     
     
         10 . The tobacco plant or part thereof or tobacco cell or cell culture according to  claim 7  wherein the content of one or more alkaloids selected from nicotine, nornicotine, PON, anabasine, myosmine, and anatabine is modulated or decreased. 
     
     
         11 . The tobacco plant or part thereof or tobacco cell or tobacco cell culture according to  claim 10 , wherein the nicotine content is decreased. 
     
     
         12 . A plant or a crop bred or grown from the tobacco plant or part thereof or tobacco cell or cell culture according to  claim 7 , the plant or the crop comprising modulated or decreased activity or expression of a FAD synthetase, or decreased alkaloid and/or TSNA precursor content in comparison to a plant or a crop bred or grown from an unmodified tobacco plant or part thereof or an unmodified tobacco cell or cell culture. 
     
     
         13 . A product or a leaf produced from the tobacco plant or part thereof or a tobacco cell or cell culture according to  claim 7 , the product or the leaf comprising modulated or decreased activity or expression of a FAD synthetase, or decreased alkaloid and/or TSNA precursor content in comparison to a product or a leaf produced from an unmodified tobacco plant or part thereof or an unmodified tobacco cell or cell culture. 
     
     
         14 - 15 . (canceled) 
     
     
         16 . A harvested leaf or a cut harvested leaf of the tobacco plant according to  claim 7 , or obtained from a plant propagated from a propagation material obtained from the tobacco plant according to  claim 7 , the harvested leaf or the cut harvested leaf comprising modulated or decreased activity or expression of a FAD synthetase, or decreased alkaloid and/or TSNA precursor content in comparison to a harvested leaf or a cut harvested leaf obtained from an unmodified tobacco plant. 
     
     
         17 . (canceled) 
     
     
         18 . A processed leaf:
 obtained by processing the tobacco plant according to  claim 7 ;   obtained from a plant or a harvested leaf thereof, wherein the plant was propagated from the plant propagation material obtained from the tobacco plant according to  claim 7 ; or   obtained by processing a harvested leaf or a cut harvested leaf of the tobacco plant according to  claim 7 , the processed leaf comprising modulated or decreased activity or expression of a FAD synthetase, or decreased alkaloid and/or TSNA precursor content in comparison to a processed leaf obtained by processing an unmodified tobacco plant or a harvested leaf or a cut harvested leaf thereof.   
     
     
         19 . The processed leaf according to  claim 18 , wherein the leaf is processed by curing, fermenting, pasteurising or a combination thereof, or wherein the leaf is a cut processed leaf. 
     
     
         20 . (canceled) 
     
     
         21 . Cured tobacco material made from the tobacco plant or a part thereof according to  claim 7 , or a harvested leaf of the tobacco plant according to  claim 7 , or a processed leaf obtained by processing the tobacco plant or part thereof according to  claim 7 , the cured tobacco material comprising modulated or decreased activity or expression of a FAD synthetase, or decreased alkaloid and/or TSNA precursor content in comparison to a cut tobacco material made from an unmodified tobacco plant, a part thereof, a harvested leaf thereof, or a processed leaf thereof. 
     
     
         22 . A tobacco blend comprising said cured tobacco material of  claim 21 . 
     
     
         23 . A tobacco industry product prepared from:
 a tobacco plant or part thereof or tobacco cell or cell culture according to  claim 7 ;   a tobacco plant or part thereof propagated from a tobacco plant propagation material obtained from the tobacco plant according to  claim 7 ;   a harvested leaf of the tobacco plant according to  claim 7 ; or   a processed leaf obtained by processing the tobacco plant according to  claim 7 ,   the tobacco industry product comprising modulated or decreased activity or expression of a FAD synthetase, or decreased alkaloid and/or TSNA precursor content in comparison to a tobacco industry product prepared from an unmodified tobacco plant or part thereof or an unmodified tobacco cell or cell culture.   
     
     
         24 . A tobacco industry product according to  claim 23 , wherein the tobacco product is a combustible smoking article, a smokeless tobacco product, or a non-combustible aerosol provision system, a tobacco heating device, or an aerosol-generating device. 
     
     
         25 - 26 . (canceled) 
     
     
         27 . A tobacco industry product comprising a plant or a part thereof according to  claim 7  or an extract thereof or a tobacco cell culture according to  claim 7 ; or a cured tobacco material made from a plant or a part thereof according to  claim 7 ; or a tobacco blend comprising the cured tobacco material, wherein the tobacco industry product comprises a combustible smoking article, a non-combustible aerosol provisioning system, a smokeless tobacco product, or a tobacco heating device, and wherein the tobacco industry product comprises modulated or decreased activity or expression of a FAD synthetase, or decreased alkaloid and/or TSNA precursor content in comparison to a tobacco industry product comprising an unmodified tobacco plant or part thereof or an extract thereof, an unmodified tobacco cell culture, or a cured tobacco material or a tobacco blend made from an unmodified plant or part thereof. 
     
     
         28 . A nucleic acid molecule comprising a mutation in a nucleotide sequence encoding a FAD synthetase protein which:
 a) encodes an amino acid sequence as set out in SEQ ID No. 3; or a functional variant or functional fragment or orthologue of SEQ ID No. 3; or a sequence which has at least 80% identity to SEQ ID No. 3; or a homologue of SEQ ID No. 3; or   b) comprises a sequence as set out in SEQ ID No. 1 or 2; or a functional variant or functional fragment or orthologue of SEQ ID No. 1 or 2; or a nucleic acid sequence which has at least 80% identity to SEQ ID No. 1 or 2; or a homologue of SEQ ID No. 1 or 2, wherein the activity or expression of the encoded FAD synthetase protein is modulated or decreased.   
     
     
         29 . A mutant plant or progeny or seed thereof comprising the nucleic acid molecule of  claim 28 , wherein the mutation comprises a heritable mutation, and
 wherein said heritable mutation modulates or decreases the activity or expression of FAD synthetase and wherein the mutant plant has modulated or decreased alkaloid content and/or modulated content of a tobacco specific nitrosamine (TSNA) or a precursor of a TSNA relative to a comparable plant which does not carry said heritable mutation.   
     
     
         30 . (canceled) 
     
     
         31 . A harvested leaf, a processed leaf, or cured tobacco material produced from the mutant plant according to  claim 29 ,
 wherein said harvested leaf, processed leaf, or cured tobacco material has modulated or decreased activity or expression of a FAD synthetase, or modulated or decreased alkaloid content and/or modulated content of a tobacco specific nitrosamine (TSNA) or a precursor of a TSNA relative to a harvested leaf, processed leaf, or cured tobacco material produced from a comparable plant which does not carry said modification in said FAD synthetase.